Kazumi Shimono

A common variant of leucine-rich repeat-containing 16A (LRRC16A) gene is associated with gout susceptibility

Gout is a common disease resulting from hyperuricemia which causes acute arthritis. Recently, genome-wide association studies revealed an association between serum uric acid levels and a common variant of leucine-rich repeat-containing 16A (LRRC16A) gene. However, it remains to be clarified whether LRRC16A contributes to the susceptibility to gout. In this study, we investigated the relationship between rs742132 in LRRC16A and gout. A total of 545 Japanese male gout cases and 1,115 male individuals as a control group were genotyped. rs742132 A/A genotype significantly increased the risk of gout, conferring an odds ratio of 1.30 (95xa0% CI 1.05–1.60; pxa0=xa00.015). LRRC16A encodes a protein called capping protein ARP2/3 and myosin-I linker (CARMIL), which serves as an inhibitor of the actin capping protein (CP). CP is an essential element of the actin cytoskeleton, which binds to the barbed end of the actin filament and regulates its polymerization. In the apical membrane of proximal tubular cells in the human kidney, the urate-transporting multimolecular complex (urate transportsome) is proposed to consist of several urate transporters and scaffolding proteins, which interact with the actin cytoskeleton. Thus, if there is a CARMIL dysfunction and regulatory disability in actin polymerization, urate transportsome may be unable to operate appropriately. We have shown for the first time that CARMIL/LRRC16A was associated with gout, which could be due to urate transportsome failure.

Converting a Light-Driven Proton Pump into a Light-Gated Proton Channel

There are two types of membrane-embedded ion transport machineries in nature. The ion pumps generate electrochemical potential by energy-coupled active ion transportation, while the ion channels produce action potential by stimulus-dependent passive ion transportation. About 80% of the amino acid residues of the light-driven proton pump archaerhodopsin-3 (AR3) and the light-gated cation channel channelrhodopsin (ChR) differ although they share the close similarity in architecture. Therefore, the question arises: How can these proteins function differently? The absorption maxima of ion pumps are red-shifted about 30-100 nm compared with ChRs, implying a structural difference in the retinal binding cavity. To modify the cavity, a blue-shifted AR3 named AR3-T was produced by replacing three residues located around the retinal (i.e., M128A, G132V, and A225T). AR3-T showed an inward H(+) flux across the membrane, raising the possibility that it works as an inward H(+) pump or an H(+) channel. Electrophysiological experiments showed that the reverse membrane potential was nearly zero, indicating light-gated ion channeling activity of AR3-T. Spectroscopic characterization of AR3-T revealed similar photochemical properties to some of ChRs, including an all-trans retinal configuration, a strong hydrogen bond between the protonated retinal Schiff base and its counterion, and a slow photocycle. From these results, we concluded that the functional determinant in the H(+) transporters is localized at the center of the membrane-spanning domain, but not in the cytoplasmic and extracellular domains.

The phenomenon of "albumin-mediated" hepatic uptake of organic anion transport polypeptide substrates: Prediction of the in vivo uptake clearance from the in vitro uptake by isolated hepatocytes using a "facilitated-dissociation" model

The effects of bovine serum albumin and human serum albumin on the unbound hepatic uptake clearance (PSu,inf) of the organic anion–transporting polypeptide substrates 1-anilino-8-naphthalene sulfonate (ANS) and pitavastatin (PTV) were determined using primary cultured rat hepatocytes and isolated human hepatocytes, respectively. The PSu,inf value of hepatocytes was estimated by dividing the initial uptake rate of these anions by their unbound concentrations. The PSu,inf values for ANS and PTV were enhanced in the presence of albumin, thereby demonstrating the phenomenon of “albumin-mediated” hepatic uptake. We previously constructed a “facilitated-dissociation” model, in which the interaction of the ligand-albumin complex with the cell surface enhanced the dissociation of that complex to provide unbound ligand for uptake to the hepatocytes [J Pharmacokinet Biopharm 16:165–181 (1988)]. That model was able to describe accurately the relationship between the enhancement of the PSu,inf values and the albumin concentration. By considering the enhancement of hepatic uptake clearance by albumin using this facilitated-dissociation model, we could predict accurately the PSu,inf in vivo from that obtained in isolated hepatocytes. In the light of these findings, we suggest that the facilitated-dissociation model is applicable to describing the phenomenon of albumin-mediated hepatic uptake via organic anion transporters and to evaluating hepatic uptake clearance in vivo.

X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION.

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a “thermal sensor.” These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum

Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.

Electrophysiological characterization of human Na⁺/taurocholate cotransporting polypeptide (hNTCP) heterologously expressed in Xenopus laevis oocytes.

The Na(+)/taurocholate cotransporting polypeptide (NTCP) plays a major role in Na(+)-dependent bile acid uptake into hepatocytes. The purpose of the present study was to establish the heterologous expression of human NTCP (hNTCP) in Xenopus laevis oocytes and to elucidate whether the transport of bile acid via hNTCP is electrogenic using electrophysiological techniques. First, we evaluated the uptake of taurocholate (TCA) by hNTCP heterologously expressed in Xenopus oocytes utilizing [(3)H]-labeled TCA. The uptake of 1.2 μM TCA by cRNA-injected oocytes increased more than 100-fold compared to H2O-injected oocytes, indicating that hNTCP is robustly expressed in the oocytes. hNTCP-mediated transport of TCA is saturable with a Michaelis constant of 10.5 ± 2.9 μM. The Na(+)-activation kinetics describing the relationship between the concentration of Na(+) and the magnitude of the TCA uptake rate by hNTCP were sigmoidal with a Hill coefficient of 2.3 ± 0.4, indicating the involvement of more than one Na(+) in the transport process. Ntcp in primary cultured hepatocytes from rats exhibited similar Na(+)-activation kinetics of TCA uptake rate with a Hill coefficient of 1.9 ± 0.1, suggesting that hNTCP could be expressed properly in the oocytes and exhibit the electrogenic property of Na(+)-coupled TCA transport. The transport of TCA via hNTCP was subsequently determined in the oocytes by the inward currents induced via TCA uptake under voltage (-50 mV). Two hundred micromolar TCA induced significant inward currents that were entirely abolished by the substitution of Na(+) with N-methyl-d-glucamine (NMDG) in the perfusate, indicating that the TCA-induced currents were obligatorily dependent on the presence of Na(+). The TCA-induced currents were saturable, and the substrate concentration needed for half-maximal induction of the current was consistent with the Michaelis constant. Transportable substrates, such as rosuvastatin and fluvastatin, also induced currents. These results in the hNTCP heterologously expressed in Xenopus oocytes directly demonstrated that hNTCP is an electrogenic Na(+)-dependent transporter.

Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry.

Halorhodopsin (HR), an inwardly directed, light-driven anion pump, is a membrane protein in halobacterial cells that contains the chromophore retinal, which binds to a specific lysine residue forming the Schiff base. An anion binds to the extracellular binding site near the Schiff base, and illumination makes this anion go to the intracellular channel, followed by its release from the protein and re-uptake from the opposite side. The thermodynamic properties of the anion binding in the dark, which have not been previously estimated, are determined using isothermal titration calorimetry (ITC). For Cl(-) as a typical substrate of HR from Natronomonas pharaonis, ΔG=-RT ln(1/K(d))=-15.9 kJ/mol, ΔH=-21.3 kJ/mol and TΔS=-5.4 kJ/mol at 35 °C, where K(d) represents the dissociation constant. In the dark, K(d) values have been determined by the usual spectroscopic methods and are in agreement with the values estimated by ITC here. Opsin showed no Cl(-) binding ability, and the deprotonated Schiff base showed weak binding affinity, suggesting the importance of the positively charged protonated Schiff base for the anion binding.

Existence of two O-like intermediates in the photocycle of Acetabularia rhodopsin II, a light-driven proton pump from a marine alga

A spectrally silent change is often observed in the photocycle of microbial rhodopsins. Here, we suggest the presence of two O intermediates in the photocycle of Acetabularia rhodopsin II (ARII or also called Ace2), a light-driven algal proton pump from Acetabularia acetabulum. ARII exhibits a photocycle including a quasi-equilibrium state of M, N, and O (M⇄N⇄O→) at near neutral and above pH values. However, acidification of the medium below pH ~5.5 causes no accumulation of N, resulting in that the photocycle of ARII can be described as an irreversible scheme (M→O→). This may facilitate the investigation of the latter part of the photocycle, especially the rise and decay of O, during which molecular events have not been sufficiently understood. Thus we analyzed the photocycle under acidic conditions (pH ≤ 5.5). Analysis of the absorbance change at 610 nm, which mainly monitors the fractional concentration changes of K and O, was performed and revealed a photocycle scheme containing two sequential O-states with the different molar extinction coefficients. These photoproducts, termed O1 and O2, may be even produced at physiological pH, although they are not clearly observed under this condition due to the existence of a long M-N-O equilibrium.

Carbidopa is an activator of aryl hydrocarbon receptor with potential for cancer therapy

Carbidopa is used with l-DOPA (l-3,4-dihydroxyphenylalanine) to treat Parkinsons disease (PD). PD patients exhibit lower incidence of most cancers including pancreatic cancer, but with the notable exception of melanoma. The decreased cancer incidence is not due to l-DOPA; however, the relevance of Carbidopa to this phenomenon has not been investigated. Here, we tested the hypothesis that Carbidopa, independent of l-DOPA, might elicit an anticancer effect. Carbidopa inhibited pancreatic cancer cell proliferation both in vitro and in vivo Based on structural similarity with phenylhydrazine, an inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1), we predicted that Carbidopa might also inhibit IDO1, thus providing a molecular basis for its anticancer effect. The inhibitory effect was confirmed using human recombinant IDO1. To demonstrate the inhibition in intact cells, AhR (aryl hydrocarbon receptor) activity was monitored as readout for IDO1-mediated generation of the endogenous AhR agonist kynurenine in pancreatic and liver cancer cells. Surprisingly, Carbidopa did not inhibit but instead potentiated AhR signaling, evident from increased CYP1A1 (cytochrome P450 family 1 subfamily A member 1), CYP1A2, and CYP1B1 expression. In pancreatic and liver cancer cells, Carbidopa promoted AhR nuclear localization. AhR antagonists blocked Carbidopa-dependent activation of AhR signaling. The inhibitory effect on pancreatic cancer cells in vitro and in vivo and the activation of AhR occurred at therapeutic concentrations of Carbidopa. Chromatin immunoprecipitation assay further confirmed that Carbidopa promoted AhR binding to its target gene CYP1A1 leading to its induction. We conclude that Carbidopa is an AhR agonist and suppresses pancreatic cancer. Hence, Carbidopa could potentially be re-purposed to treat pancreatic cancer and possibly other cancers as well.

Production of a Light-Gated Proton Channel by Replacing the Retinal Chromophore with Its Synthetic Vinylene Derivative

Rhodopsin is widely distributed in organisms as a membrane-embedded photoreceptor protein, consisting of the apoprotein opsin and vitamin-A aldehyde retinal, A1-retinal and A2-retinal being the natural chromophores. Modifications of opsin (e.g., by mutations) have provided insight into the molecular mechanism of the light-induced functions of rhodopsins as well as providing tools in chemical biology to control cellular activity by light. Instead of the apoprotein opsin, in this study, we focused on the retinal chromophore and synthesized three vinylene derivatives of A2-retinal. One of them, C(14)-vinylene A2-retinal (14V-A2), was successfully incorporated into the opsin of a light-driven proton pump archaerhodopsin-3 (AR3). Electrophysiological experiments revealed that the opsin of AR3 (archaeopsin3, AO3) with 14V-A2 functions as a light-gated proton channel. The engineered proton channel showed characteristic photochemical properties, which are significantly different from those of AR3. Thus, we successfully produced a proton channel by replacing the chromophore of AR3.